TP53, a well-known tumor suppressor gene, is frequently inactivated by mutation or deletion in a majority of human tumors. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment due to the complexity of p53 signaling. Therefore, identification of vulnerabilities conferred by TP53 deletion or mutation is a major challenge to target p53 aberrancy in human cancer. We demonstrate that genomic deletion of TP53 frequently encompasses neighboring essential genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is always co-deleted with TP53 in human cancers. Hemizygous loss of TP53/POLR2A occurs in 53% of colorectal cancers (CRC), 62% of breast cancers, 75% of ovarian cancers, and 41% of pancreatic cancers. POLR2A encodes the largest and catalytic subunit of RNA polymerase II complex. It is specifically inhibited by ?-Amanitin, a cyclic 8-aa peptide toxin found in the death cap mushroom (Amanita phalloides). POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer (CRC). Suppression of POLR2A selectively inhibits proliferation, survival and tumorigenic potential of CRC cells with hemizygous TP53 loss in a p53- independent manner. Previous clinical applications of ?-Amanitin have been limited due to its liver toxicity. Free ?-Amanitin causes apoptosis and necrosis of hepatocytes by interacting with the hepatocyte-specific transporting protein OATP1B3 (10). However, ?-Amanitin is no longer substrate for OATP1B3 when coupled to antibodies. Therefore, ?-Amanitin-based antibody drug conjugates (ADCs) are highly effective therapeutic agents with significantly reduced toxicity. Our study has shown that low doses of ?-Amanitin-conjugated anti- EpCAM (Epithelial Cell Adhesion Molecule) antibody lead to complete tumor regression in murine models of human CRC with hemizygous deletion of POLR2A. The preclinical studies in this application were recently published in Nature, which provide the foundation for future clinical trials. There is already discussion regarding the potential for initiation of a clinical trial based on our work. We propose that hemizygous deletion of TP53 in human cancers creates a therapeutic window between normal and cancer cells, and that PORL2A is a novel and druggable target in cancers with such genomic alterations.